Members of the G-protein coupled receptor (GPCR) class of membrane proteins (also known as seven-transmembrane spanning or 7TM receptors and serpentine receptors) mediate cellular signalling in response to a very wide variety of extracellular signals, is including hormones, neurotransmitters, cytokines and even environmental substances such as odours and tastes. In response to the ligand interacting with the extracellular portion of the receptor (most usually the N-terminal tail of the receptor protein), the receptor is converted temporaily to an activated state (this conversion is usually designated R+L→R*L where R is the inactive receptor, R* is the activated receptor and L is the ligand).
The activated (or R*) conformation of the receptor is then able to interact with a member of the G-protein family. The G-proteins are a large family of trimeric intracellular proteins which bind guanine nucleotides. On interacting with the activated receptor (probably by a mechanism called “collisional coupling”) the G-protein exchanges a bound guanosine diphosphate (GDP) for a guanosine triphosphate (GTP). In this GTP-bound form the G-protein trimer dissociates, yielding a free Gα subunit, and a βγ dimer. Both the Gα and βγ subunits can then participate in further signalling cascades. For example, the Gα subunit can activate the adenylate cyclase (AC) enzyme, which generates cyclic adenosine monophospate (cAMP) from adenosine triphosphate. The βγ subunit can activate members of the PI-3-kinase family of enzymes. Ultimately, these signals can result in modulation of almost every aspect of cell behaviour, from contraction to motility, metabolism to further signalling.
The signal, once activated, is then slowly turned off by a number of mechanisms. The GTP associated with the Gα subunit is hydrolysed back to GDP, resulting in the reassociation of the Gα and βγ subunits to form the inactive trimeric GDP-bound G-m protein. The GPCR itself also becomes phosphorylated on the intracellular C-terminus, preventing further interaction with G-proteins. Eventually, the bound ligand may also dissociate.
This generic signalling pathway is so central and ubiquitous in mammalian physiology that as many as 40% of licensed pharmaceuticals have a GPCR among their molecular is targets. Similarly, bacteria have evolved to target G-protein signalling in order to disrupt host physiology and immunity: Vibrio cholerae (the organism responsible for cholera), for example, makes a protein known as cholera toxin which irreversibly inhibits the Gα subunit of a widely distributed G-protein called Gs. Similarly, Bordetella pertussis (the organism responsible for Whooping Cough) makes a protein known as Pertussis toxin which has a similar effect on a different G-protein, Gi.
One approach to identifying pharmaceuticals which will modulate GPCR signalling has been to screen very large random compound libraries for the ability to interfere with ligand binding to membrane preparations containing recombinant or purified GPCRs. In such high throughput screens, various methods have been adopted to facilitate the detection of binding. For example, in scintillation proximity assays, the binding of a radiolabelled ligand to the receptor brings the radionuclide into proximity with a scintillant molecule bound to the receptor—as the nuclide decays, light is emitted which can be detected and quantified. Alternatively, the ligand can be fluorescently labelled and the binding detected by fluorescence polarisation (dependent on the reduced rotational degress of freedom of the fluorescent tag when the ligand is immobilised on binding to the receptor).
While these techniques have been successful in some instances, and yielded lead compounds which have subsequently been developed as human pharmaceuticals (for example, the 5HT3 receptor antagonist Ondansetron, used to treat migraine headaches), there remain large numbers of GPCRs for which few, if any, suitable non-peptide agonist or antagonist compounds have been identified, despite intensive screening across the pharmaceutical industry. For example, there are few specific non-peptide antagonists for the chemokine receptor family of GPCRs, and no agonists. Since chemokines play a central role in immune regulation, such molecules would be expected to be extremely valuable pharmaceuticals with immunomodulatory properties useful in treating a wide range of diseases with an inflammatory component.
Two factors limit the likely success of random screening programmes: firstly, there is a very large compound space to be screened, and even with the best available high throughput technology and the best combinatorial chemistry approaches to generating diverse libraries, only a small fraction of all possible molecular structures can be investigated. Secondly, even when leads have been successfully identified the core pharmacophores are often not suitable for use in vivo—the lead compound and its analogs may be simply too toxic.
Another major problem with such “negative screening” paradigms (where you detect the ability of the test library to block binding of a labelled ligand) is that most of the leads identified are receptor antagonists. Few of the leads have any agonist activity (as expected—agonist activity requires the ability to bind to and then convert the receptor to the activated conformation, whereas antagonist actively merely requires the ability to bind to the receptor or ligand in such a way as to prevent their interactions) and generating analogs of the initial antagonist leads to convert them to agonists is a “hit and miss” affair with very low success rates.
One approach to circumventing this problem would be to replace the random compound library with a library of molecular structures preselected to contain a high proportion of GPCR binding compounds. Such a library would also ideally include both agonists and antagonists in similar proportion so that either could be readily located. Ideally, also, the basic molecular structures used in the library would be non-toxic.
Whether or nor real libraries can be constructed which approximate these ideal properties is not at all clear. If they do, it will require the existance of a putative “ideal” GPCR substrate which would interact with many different GPCRs irrespective of their natural ligand preferences. By varying the substitution of this idealised substrate it may then be possible to impart selectivity for one receptor in the class over all the others.
Here we describe an “ideal” GPCR substrate which can be used as a three-dimensional skeleton that can be variously substituted to generate agonists and/or anatagonists at a range of different GPCRs. The invention also provides for the preparation of a library of said substituted compounds to be applied in a screening process in order to generate GPCR ligands with any prescribed set of specificities. In this way, it is now possible to “dial up” a GPCR ligand with a known set of properties (for example, a ligand which has agonist activity at dopamine D2 receptors at the same time as antagonist activity at serotonin 5HT1a (receptors). In contrast, identifying such mixed ligands serendipitously from random libraries is a very rare event.